Process for preparation of canola protein isolates

ABSTRACT

A novel canola protein isolate having predominantly of 2S canola protein and having improved solubility properties, has an increased proportion of 2S canola protein and a decreased proportion of 7S canola protein. The novel canola protein isolate is formed by heat treatment of aqueous supernatant from canola protein micelle formation and precipitation, to effect precipitation of 7S protein which is sedimented and removed. Alternatively, the novel canola protein isolate may be derived from a selective membrane procedure in which an aqueous canola protein solution containing 12S, 7S and 2S canola proteins is subjected to a first selective membrane technique to retain 12S and 7S canola proteins in a retentate, which is dried to provide a canola protein isolate consisting predominantly of 7S canola protein, and to permit 2S canola protein to pass through the membrane, the permeate is subjected to a second selective membrane technique to retain 2S canola protein and to permit low molecular weight contaminants to pass through the membrane, and the retentate from the latter membrane technique is dried.

REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.11/038,086 filed Jan. 21, 2005, which claims priority under 35 USC119(e) from U.S. Provisional Patent Application No. 60/537,031 filedJan. 20, 2004.

FIELD OF INVENTION

The present invention relates to the production of canola proteinisolates.

BACKGROUND TO THE INVENTION

Canola oil seed protein isolates having protein contents of at least 100wt % (N×6.25) can be formed from oil seed meal by a process as describedin copending U.S. patent application Ser. No. 10/137,391 filed May 3,2002 (WO 02/089597), assigned to the assignee hereof and the disclosuresof which are incorporated herein by reference. The procedure involves amultiple step process comprising extracting canola oil seed meal using asalt solution, separating the resulting aqueous protein solution fromresidual oil seed meal, increasing the protein concentration of theaqueous solution to at least about 200 g/L while maintaining the ionicstrength substantially constant by using a selective membrane technique,diluting the resulting concentrated protein solution into chilled waterto cause the formation of protein micelles, settling the proteinmicelles to form an amorphous, sticky, gelatinous, gluten-like proteinmicellar mass (PMM), and recovering the protein micellar mass fromsupernatant having a protein content of at least about 100 wt %(N×6.25). As used herein, protein content is determined on a dry weightbasis. The recovered PMM may be dried.

In one embodiment of the process, the supernatant from the PMM settlingstep is processed to recover canola protein isolate from thesupernatant. This procedure may be effected by initially concentratingthe supernatant using an ultrafiltration membrane and drying theconcentrate. The resulting canola protein isolate has a protein contentof at least about 90 wt %, preferably at least about 100 wt % (N×6.25).

The procedures described in U.S. patent application Ser. No. 10/137,391are essentially batch procedures. In copending U.S. patent applicationSer. No. 10/298,678 filed Nov. 19, 2002 (WO 03/043439), assigned to theassignee hereof and the disclosures of which are incorporated herein byreference, there is described a continuous process for making canolaprotein isolates. In accordance therewith, canola oil seed meal iscontinuously mixed with a salt solution, the mixture is conveyed througha pipe while extracting protein from the canola oil seed meal to form anaqueous protein solution, the aqueous protein solution is continuouslyconveyed through a selective membrane operation to increase the proteincontent of the aqueous protein solution to at least about 50 g/L, whilemaintaining the ionic strength substantially constant, the resultingconcentrated protein solution is continuously mixed with chilled waterto cause the formation of protein micelles, and the protein micelles arecontinuously permitted to settle while the supernatant is continuouslyoverflowed until the desired amount of PMM has accumulated in thesettling vessel. The PMM is recovered from the settling vessel and maybe dried. The PMM has a protein content of at least about 90 wt %(N×6.25), preferably at least about 100 wt %. The overflowed supernatantmay be processed to recover canola protein isolate therefrom, asdescribed above.

Canola seed is known to contain about 10 to about 30 wt % proteins andseveral different protein components have been identified. Theseproteins include a 12S globulin, known as cruciferin, a 7S protein and a2S storage protein, known as napin. As described in copending U.S.patent application Ser. No. 10/413,371 filed Apr. 15, 2003 (WO03/088760), assigned to the assignee hereof and the disclosures of whichare incorporated herein by reference, the procedures described above,involving dilution of concentrated aqueous protein solution to form PMMand processing of supernatant to recover additional protein, lead to therecovery of isolates of different protein profiles.

In this regard, the PMM-derived canola protein isolate has a proteincomponent content of about 60 to about 98 wt % of 7S protein, about 1 toabout 15 wt % of 12S protein and 0 to about 25 wt % of 2S protein. Thesupematant-derived canola protein isolate has a protein componentcontent of about 60 to about 95 wt % of 2S protein, about 5 to about 40wt % of 7S protein and 0 to about 5 wt % of 12S protein. Thus, thePMM-derived canola protein isolate is predominantly 7S protein and thesupematant-derived canola protein isolate is predominantly 2S protein.As described in the aforementioned U.S. patent application Ser. No.10/413,371, the 2S protein has a molecular size of about 14,000 daltons,the 7S protein has a molecular mass of about 145,000 daltons and the 12Sprotein has a molecular size of about 290,000 daltons.

Canola is also known as rapeseed or oil seed rape.

SUMMARY OF INVENTION

It has now surprisingly been found that a novel canola protein isolatehaving an increased proportion of 2S protein, preferably containing atleast about 85 wt % of 2S protein, and having a reduced proportion of 7Sprotein exhibits superior properties in aqueous solution to thesupematant-derived canola protein isolate prepared following theprocedure of the aforementioned U.S. patent application Ser. No.10/137,391.

In addition to improved solubility at a variety of pH values, the novelcanola protein isolate provided herein is able to provide improvedclarity in solution with soft drinks, providing clear protein fortifiedsoft drinks.

Accordingly, in one aspect of the present invention, there is provided acanola protein isolate consisting predominantly of 2S canola proteinhaving a protein content of at least about 90 wt % of (N×6.25) on a dryweight basis (d.b.) and having an increased proportion of 2S canolaprotein and a decreased proportion of 7S canola protein when compared tocanola protein isolates consisting predominantly of 2S canola proteinand derived from aqueous supernatant from canola protein micelleformation and precipitation.

In a further aspect of the present invention, there is provided a canolaprotein isolate having a protein content of at least about 90 wt %(N×6.25) on a dry weight basis (d.b.) and containing at least about 85wt % of 2S canola protein and less than about 15 wt % of 7S canolaprotein of the canola proteins present in the isolate.

The novel canola protein isolate may be prepared by thermal treatment ofthe concentrated supernatant from the procedure of U.S. patentapplication Ser. No. 10/137,391 in order to reduce the proportion of 7Sprotein in the concentrated supernatant and hence to increase theproportion of 2S protein. Accordingly, in another aspect of the presentinvention, there is provided a process for the preparation of a canolaprotein isolate having an increased proportion of 2S canola protein,which comprises (a) providing an aqueous solution of 2S and 7S proteinsconsisting predominantly of 2S protein, (b) heat treating the aqueoussolution to cause precipitation of 7S canola protein, (c) removingprecipitated 7S protein from the aqueous solution, and (d) recovering acanola protein isolate having a protein content of at least about 90 wt% (N×6.25) d.b. and having an increased proportion of 2S canola protein.

Alternatively, the novel canola protein isolate may be prepared by aprocedure in which, following extraction of protein from the canola oilseed meal, the protein solution is subjected to a first selectivemembrane step with a membrane having a molecular weight cut-off whichpermits the 2S protein to pass through the membrane in a permeate whilethe 7S and 12S proteins are retained in a retentate. The retentate thenis dried to provide a first canola protein isolate which ispredominantly 7S protein. The permeate from the first selective membraneprocess step is then subjected to a second selective membrane step witha membrane having a molecular weight cut-off which retains the 2Sprotein and permits low molecular weight contaminants, including salt,phenolics and anti-nutritional materials, to pass through. The retentatefrom the latter selective membrane step then is dried to provide asecond canola protein isolate which is predominantly 2S protein andwhich is the novel protein isolate.

Accordingly, in an additional aspect of the present invention, there isprovided a process for the preparation of a canola protein isolate,which comprises (a) providing an aqueous canola protein solution derivedfrom canola oil seed meal and containing 12S, 7S and 2S canola proteins,(b) increasing the protein concentration of the aqueous solution using aselective membrane technique which is effective to retain 7S and 12Scanola proteins in a retentate and to permit 2S protein to pass throughthe membrane as a permeate to provide a concentrated protein solution,(c) drying the retentate from step (b) to provide a canola proteinisolate consisting predominantly of 7S canola protein and having aprotein content of at least about 90 wt % (N×6.25) on a dry weight basis(d.b.), (d) increasing the concentration of the permeate from step (a)using a selective membrane technique which is effective to retain 2Scanola protein in a retentate and to permit low molecular weightcontaminants to pass through the membrane in a permeate, and (e) dryingthe retentate from step (d) to provide a canola protein isolateconsisting predominantly of 2S protein and having a protein content ofat least about 90 wt % (N×6.25) d.b.

BRIEF DESCRIPTION OF DRAWING

FIG. 1 is a schematic representation of a protein solution recoveryprocess according to one embodiment of the invention superimposed on theprotein micelle process.

GENERAL DESCRIPTION OF INVENTION

The novel canola protein isolate provided herein has a protein contentof at least about 90 wt % (N×6.25), preferably at least about 100 wt %,and may be isolated from canola oil seed meal by a batch process, or acontinuous process, or a semi-continuous process.

The novel canola protein isolate provided herein consists predominantlyof 2S protein and has an increased proportion of 2S canola protein and adecreased proportion of 7S canola protein when compared to canolaprotein isolates consisting predominantly of 2S protein and derived fromsupernatant from canola protein micelle formation and precipitation andprepared under the same experimental conditions of preparation.

The novel canola protein isolates contain at least about 85 wt % of 2Scanola protein and less than about 15 wt % of 7S canola protein,preferably at least about 90 wt % of 2S canola protein and less thanabout 10 wt % of 7S canola protein and more preferably as great aproportion of 2S protein as is possible. As noted above, such canolaprotein isolate may be obtained by heat treatment of concentratedsupernatant, as described in more detail below. The heat treatment ofthe concentrated supernatant causes precipitation of the 7S protein,which can be removed from the heat-treated supernatant by any convenientmeans, such as centrifugation. The 2S protein is not affected by theheat treatment and hence the heat treatment increases the proportion of2S protein present by decreasing the proportion of 7S protein.

The novel canola protein isolate is soluble in aqueous solution over awide range of pH values, generally having greater solubility than canolaprotein isolate consisting predominantly of 2S protein and derived fromsupernatant from canola protein micelle formation and precipitationunder the same experimental conditions of preparation. In addition,aqueous solutions of the novel canola protein isolate in soft drinks,including carbonated soft drinks, such as those commercially-available,have a greater clarity than such aqueous solutions produced from canolaprotein isolate consisting predominantly of 2S protein and derived fromsupernatant from canola protein micelle formation and precipitationunder the same conditions of preparation.

The concentration of canola protein isolate in the aqueous solution,including solution in soft drinks, may vary depending on the intendeduse of the solution. In general, the protein concentration may vary fromabout 0.1 to about 30 wt %, preferably about 1 to about 5 wt %.

The initial step of the process of providing canola protein isolatesinvolves solubilizing proteinaceous material from canola oil seed meal.The proteinaceous material recovered from canola seed meal may be theprotein naturally occurring in canola seed or the proteinaceous materialmay be a protein modified by genetic manipulation but possessingcharacteristic hydrophobic and polar properties of the natural protein.The canola meal may be any canola meal resulting from the removal ofcanola oil from canola oil seed with varying levels of non-denaturedprotein, resulting, for example, from hot hexane extraction or cold oilextrusion methods. The removal of canola oil from canola oil seedusually is effected as a separate operation from the protein isolaterecovery procedure described herein.

Protein solubilization is effected most efficiently by using a foodgrade salt solution since the presence of the salt enhances the removalof soluble protein from the oil seed meal. Where the canola proteinisolate is intended for non-food uses, non-food-grade chemicals may beused. The salt usually is sodium chloride, although other salts, suchas, potassium chloride, may be used. The salt solution has an ionicstrength of at least about 0.05, preferably at least about 0.10, toenable solubilization of significant quantities of protein to beeffected. As the ionic strength of the salt solution increases, thedegree of solubilization of protein in the oil seed meal initiallyincreases until a maximum value is achieved. Any subsequent increase inionic strength does not increase the total protein solubilized. Theionic strength of the food grade salt solution which causes maximumprotein solubilization varies depending on the salt concerned and theoil seed meal chosen.

In view of the greater degree of dilution required for proteinprecipitation with increasing ionic strengths, it is usually preferredto utilize an ionic strength value less than about 0.8, and morepreferably a value of about 0.1 to about 0.15.

In a batch process, the salt solubilization of the protein is effectedat a temperature of at least about 5° C. and preferably up to about 35°C., preferably accompanied by agitation to decrease the solubilizationtime, which is usually about 10 to about 60 minutes. It is preferred toeffect the solubilization to extract substantially as much protein fromthe oil seed meal as is practicable, so as to provide an overall highproduct yield.

The lower temperature limit of about 5° C. is chosen sincesolubilization is impractically slow below this temperature while theupper preferred temperature limit of about 35° C. is chosen since theprocess becomes uneconomic at higher temperature levels in a batch mode.

In a continuous process, the extraction of the protein from the canolaoil seed meal is carried out in any manner consistent with effecting acontinuous extraction of protein from the canola oil seed meal. In oneembodiment, the canola oil seed meal is continuously mixed with a foodgrade salt solution and the mixture is conveyed through a pipe orconduit having a length and at a flow rate for a residence timesufficient to effect the desired extraction, in accordance with theparameters described herein. In such continuous procedure, the saltsolubilization step is effected rapidly, in a time of up to about 10minutes, preferably to effect solubilization to extract substantially asmuch protein from the canola oil seed meal as is practicable. Thesolubilization in the continuous procedure preferably is effected atelevated temperatures, preferably at least about 35° C., generally up toabout 65° C. or more.

The aqueous food grade salt solution generally has a pH of about 5 toabout 6.8, preferably about 5.3 to about 6.2, the pH of the saltsolution may be adjusted to any desired value within the range of about5 to about 6.8 for use in the extraction step by the use of anyconvenient acid, usually hydrochloric acid, or alkali, usually sodiumhydroxide, as required.

The concentration of oil seed meal in the food grade salt solutionduring the solubilization step may vary widely. Typical concentrationvalues are about 5 to about 15% w/v.

The protein extraction step with the aqueous salt solution has theadditional effect of solubilizing fats which may be present in thecanola meal, which then results in the fats being present in the aqueousphase.

The protein solution resulting from the extraction step generally has aprotein concentration of about 5 to about 40 g/L, preferably about 10 toabout 30 g/L.

The aqueous salt solution may contain an antioxidant. The antioxidantmay be any convenient antioxidant, such as sodium sulfite or ascorbicacid. The quantity of antioxidant employed may vary from about 0.01 toabout 1 wt % of the solution, preferably about 0.05 wt %. Theantioxidant serves to inhibit oxidation of phenolics in the proteinsolution.

The aqueous phase resulting from the extraction step then may beseparated from the residual canola meal, in any convenient manner, suchas by employing a decanter centrifuge, followed by disc centrifugationand/or filtration to remove residual meal. The separated residual mealmay be dried for disposal.

The colour of the final canola protein isolate can be improved in termsof light colour and less intense yellow by the mixing of powderedactivated carbon or other pigment adsorbing agent with the separatedaqueous protein solution and subsequently removing the adsorbent,conveniently by filtration, to provide a protein solution. Diafiltrationalso may be used for pigment removal.

Such pigment removal step may be carried out under any convenientconditions, generally at the ambient temperature of the separatedaqueous protein solution, employing any suitable pigment adsorbingagent. For powdered activated carbon, an amount of about 0.025% to about5% w/v, preferably about 0.05% to about 2% w/v, is employed.

Where the canola seed meal contains significant quantities of fat, asdescribed in U.S. Pat. Nos. 5,844,086 and 6,005,076, assigned to theassignee hereof and the disclosures of which are incorporated herein byreference, then the defatting steps described therein may be effected onthe separated aqueous protein solution and on the concentrated aqueousprotein solution discussed below. When the colour improvement step iscarried out, such step may be effected after the first defatting step.

As an alternative to extracting the oil seed meal with an aqueous saltsolution, such extraction may be made using water alone, although theutilization of water alone tends to extract less protein from the oilseed meal than the aqueous salt solution. Where such alternative isemployed, then the salt, in the concentrations discussed above, may beadded to the protein solution after separation from the residual oilseed meal in order to maintain the protein in solution during theconcentration step described below. When a first fat removal step iscarried out, the salt generally is added after completion of suchoperations.

Another alternative procedure is to extract the oil seed meal with thefood grade salt solution at a relatively high pH value above about 6.8,generally up to about 9.9. The pH of the food grade salt solution, maybe adjusted in pH to the desired alkaline value by the use of anyconvenient food-grade alkali, such as aqueous sodium hydroxide solution.Alternatively, the oil seed meal may be extracted with the salt solutionat a relatively low pH below about pH 5, generally down to about pH 3.Where such alternative is employed, the aqueous phase resulting from theoil seed meal extraction step then is separated from the residual canolameal, in any convenient manner, such as by employing decantercentrifugation, followed by disc centrifugation and/or filtration toremove residual meal. The separated residual meal may be dried fordisposal.

The aqueous protein solution resulting from the high or low pHextraction step then is pH adjusted to the range of about 5 to about6.8, preferably about 5.3 to about 6.2, as discussed above, prior tofurther processing as discussed below. Such pH adjustment may beeffected using any convenient acid, such as hydrochloric acid, oralkali, such as sodium hydroxide, as appropriate.

The aqueous protein solution may be processed in two alternativeprocedures, depending on whether 7S-rich protein micellar mass is to beprecipitated to leave a supernatant for processing to form the novelcanola protein isolate, or the aqueous protein solution is to beprocessed by a two-membrane operation without precipitation of proteinmicellar mass to obtain the novel canola protein isolate.

In the first alternative procedure, the aqueous protein solution isconcentrated to increase the protein concentration thereof whilemaintaining the ionic strength thereof substantially constant. Suchconcentration generally is effected to provide a concentrated proteinsolution having a protein concentration of at least about 50 g/L,preferably at least about 200 g/L, more preferably at least about 250g/L.

The concentration step may be effected in any convenient mannerconsistent with batch or continuous operation, such as by employing anyconvenient selective membrane technique, such as ultrafiltration ordiafiltration, using membranes, such as hollow-fibre membranes orspiral-wound membranes, with a suitable molecular weight cut-off, suchas about 3,000 to about 100,000 daltons, preferably about 5,000 to about10,000 daltons, having regard to differing membrane materials andconfigurations, and, for continuous operation, dimensioned to permit thedesired degree of concentration as the aqueous protein solution passesthrough the membranes.

The concentrated protein solution then may be subjected to adiafiltration step using an aqueous salt solution of the same molarityand pH as the extraction solution. Such diafiltration may be effectedusing from about 2 to about 20 volumes of diafiltration solution,preferably about 5 to about 10 volumes of diafiltration solution. In thediafiltration operation, further quantities of contaminants are removedfrom the aqueous protein solution by passage through the membrane withthe permeate. The diafiltration operation may be effected until nosignificant further quantities of phenolics and visible colour arepresent in the permeate. Such diafiltration may be effected using thesame membrane as for the concentration step. However, if desired, thediafiltration step may be effected using a separate membrane with adifferent molecular weight cut-off, such as a membrane having amolecular weight cut-off in the range of about 3,000 to about 100,000daltons, preferably about 5,000 to about 10,000 daltons, having regardto different membrane materials and configuration.

An antioxidant may be present in the diafiltration medium during atleast part of the diaflitration step. The antioxidant may be anyconvenient antioxidant, such as sodium sulfite or ascorbic acid. Thequantity of antioxidant employed in the diafiltration medium depends onthe materials employed and may vary from about 0.01 to about 1 wt %,preferably about 0.05 wt %. The antioxidant serves to inhibit oxidationof phenolics present in the concentrated canola protein isolatesolution.

The concentration step and the diafiltration step may be effected at anyconvenient temperature, generally about 20 to about 60° C., preferablyabout 20 to about 30° C., and for the period of time to effect thedesired degree of concentration. The temperature and other conditionsused to some degree depend upon the membrane equipment used to effectthe concentration and the desired protein concentration of the solution.

The concentrating of the protein solution to the preferred concentrationabove about 200 g/L in this step not only increases the process yield tolevels above about 40% in terms of the proportion of extracted proteinwhich is recovered as dried protein isolate, preferably above about 80%,but also decreases the salt concentration of the final protein isolateafter drying. The ability to control the salt concentration of theisolate is important in applications of the isolate where variations insalt concentrations affect the functional and sensory properties in aspecific food application.

As is well known, ultrafiltration and similar selective membranetechniques permit low molecular weight species to pass therethroughwhile preventing higher molecular weight species from so doing. The lowmolecular weight species include not only the ionic species of the foodgrade salt but also low molecular weight materials extracted from thesource material, such as, carbohydrates, pigments and anti-nutritionalfactors, as well as any low molecular weight forms of the protein. Themolecular weight cut-off of the membrane is usually chosen to ensureretention of a significant proportion of the protein in the solution,while permitting contaminants to pass through having regard to thedifferent membrane materials and configurations.

The concentrated and optionally diafiltered protein solution may besubject to a further defatting operation, if required, as described inU.S. Pat. Nos. 5,844,086 and 6,005,076.

The concentrated and optionally diafiltered protein solution may besubject to a colour removal operation as an alternative to the colourremoval operation described above. Powdered activated carbon may be usedherein as well as granulated activated carbon (GAC). Another materialwhich may be used as a colour absorbing agent is polyvinyl pyrrolidone.

The colour absorbing agent treatment step may be carried out under anyconvenient conditions, generally at the ambient temperature of thecanola protein solution. For powdered activated carbon, an amount ofabout 0.025% to about 5% w/v, preferably about 0.05% to about 2% w/v,may be used. Where polyvinylpyrrolidone is used as the colour absorbingagent, an amount of about 0.5% to about 5% w/v, preferably about 2% toabout 3% w/v, may be used. The colour absorbing agent may be removedfrom the canola protein solution by any convenient means, such as byfiltration.

The concentrated and optionally diafiltered protein solution resultingfrom the optional colour removal step may be subjected to pasteurizationto kill any bacteria which may have been present in the original meal asa result of storage or otherwise and extracted from the meal into thecanola protein isolate solution in the extraction step. Suchpasteurization may be effected under any desired pasteurizationconditions. Generally, the concentrated and optionally diafilteredprotein solution is heated to a temperature of about 55° to about 70°C., preferably about 60° to about 65° C., for about 10 to about 15minutes, preferably about 10 minutes. The pasteurized concentratedprotein solution then may be cooled for further processing as describedbelow, preferably to a temperature of about 25° to about 40° C.

Depending on the temperature employed in the concentration step andoptional diafiltration step and whether or not a pasteurization step iseffected, the concentrated protein solution may be warmed to atemperature of at least about 20°, and up to about 60° C., preferablyabout 25° to about 40° C., to decrease the viscosity of the concentratedprotein solution to facilitate performance of the subsequent dilutionstep and micelle formation. The concentrated protein solution should notbe heated beyond a temperature above which micelle formation does notoccur on dilution by chilled water.

The concentrated protein solution resulting from the concentration step,and optional diafiltration step, optional colour removal step, optionalpasteurization step and optional defatting step, then is diluted toeffect micelle formation by mixing the concentrated protein solutionwith chilled water having the volume required to achieve the degree ofdilution desired. Depending on the proportion of canola protein desiredto be obtained by the micelle route and the proportion from thesupernatant, the degree of dilution of the concentrated protein solutionmay be varied. With higher dilution levels, in general, a greaterproportion of the canola protein remains in the aqueous phase.

When it is desired to provide the greatest proportion of the protein bythe micelle route, the concentrated protein solution is diluted by about15 fold or less, preferably about 10 fold or less.

The chilled water with which the concentrated protein solution is mixedhas a temperature of less than about 15° C., generally about 3° to about15° C., preferably less than about 10° C., since improved yields ofprotein isolate in the form of protein micellar mass are attained withthese colder temperatures at the dilution factors used.

In a batch operation, the batch of concentrated protein solution isadded to a static body of chilled water having the desired volume, asdiscussed above. The dilution of the concentrated protein solution andconsequential decrease in ionic strength causes the formation of acloud-like mass of highly associated protein molecules in the form ofdiscrete protein droplets in micellar form. In the batch procedure, theprotein micelles are allowed to settle in the body of chilled water toform an aggregated, coalesced, dense, amorphous sticky gluten-likeprotein micellar mass (PMM). The settling may be assisted, such as bycentrifugation. Such induced settling decreases the liquid content ofthe protein micellar mass, thereby decreasing the moisture contentgenerally from about 70% by weight to about 95% by weight to a value ofgenerally about 50% by weight to about 80% by weight of the totalmicellar mass. Decreasing the moisture content of the micellar mass inthis way also decreases the occluded salt content of the micellar mass,and hence the salt content of dried isolate.

Alternatively, the dilution operation may be carried out continuously bycontinuously passing the concentrated protein solution to one inlet of aT-shaped pipe, while the diluting water is fed to the other inlet of theT-shaped pipe, permitting mixing in the pipe. The diluting water is fedinto the T-shaped pipe at a rate sufficient to achieve the desireddegree of dilution of the concentrated protein solution.

The mixing of the concentrated protein solution and the diluting waterin the pipe initiates the formation of protein micelles and the mixtureis continuously fed from the outlet from the T-shaped pipe into asettling vessel, from which, when full, supernatant is permitted tooverflow. The mixture preferably is fed into the body of liquid in thesettling vessel in a manner which minimizes turbulence within the bodyof liquid.

In the continuous procedure, the protein micelles are allowed to settlein the settling vessel to form an aggregated, coalesced, dense,amorphous, sticky, gluten-like protein micellar mass (PMM) and theprocedure is continued until a desired quantity of the PMM hasaccumulated in the bottom of the settling vessel, whereupon theaccumulated PMM is removed from the settling vessel. In lieu of settlingby sedimentation, the PMM may be separated continuously bycentrifugation.

The combination of process parameters of concentrating of the proteinsolution to a preferred protein content of at least about 200 g/L andthe use of a dilution factor less than about 15, result in higheryields, often significantly higher yields, in terms of recovery ofprotein in the form of protein micellar mass from the original mealextract, and much purer isolates in terms of protein content thanachieved using any of the known prior art protein isolate formingprocedures discussed in the aforementioned U.S. patents.

By the utilization of a continuous process for the recovery of canolaprotein isolate as compared to the batch process, the initial proteinextraction step can be significantly reduced in time for the same levelof protein extraction and significantly higher temperatures can beemployed in the extraction step. In addition, in a continuous operation,there is less chance of contamination than in a batch procedure, leadingto higher product quality and the process can be carried out in morecompact equipment.

The settled isolate is separated from the residual aqueous phase orsupernatant, such as by decantation of the residual aqueous phase fromthe settled mass or by centrifugation. The PMM may be used in the wetform or may be dried, by any convenient technique, such as spray dryingor freeze drying, to a dry form. The dry PMM has a high protein content,in excess of about 90 wt % protein, preferably at least about 100 wt %protein (calculated as Kjeldahl N×6.25), and is substantiallyundenatured (as determined by differential scanning calorimetry). Thedry PMM isolated from fatty oil seed meal also has a low residual fatcontent, when the procedures of U.S. Pat. Nos. 5,844,086 and 6,005,076are employed as necessary, which may be below about 1 wt %.

As described in the aforementioned U.S. patent application Ser. No.10/413,371, the PMM consists predominantly of a 7S canola protein havinga protein component content of about 60 to 98 wt % of 7S protein, about1 to about 15 wt % of 12S protein and 0 to about 25 wt % of 2S protein.

The supernatant from the PMM formation and settling step containssignificant amounts of canola protein, not precipitated in the dilutionstep, and is processed to recover canola protein isolate therefrom. Asdescribed in the aforementioned U.S. patent application Ser. No.10/413,371, the canola protein isolate derived from the supernatantconsists predominantly of 2S canola protein having a protein componentcontent of about 60 to about 95 wt % of 2S protein, about 5 to about 40wt % of a 7S protein and 0 to about 5 wt % of 12S protein.

The supernatant from the dilution step, following removal of the PMM, isconcentrated to increase the protein concentration thereof. Suchconcentration is effected using any convenient selective membranetechnique, such as ultrafiltration, using membranes with a suitablemolecular weight cut-off permitting low molecular weight species,including the salt and other non-proteinaceous low molecular weightmaterials extracted from the protein source material, to pass throughthe membrane, while retaining canola protein in the solution.Ultrafiltration membranes having a molecular weight cut-off of about3,000 to 100,000 daltons, preferably about 5,000 to about 10,000daltons, having regard to differing membrane materials andconfiguration, may be used. Concentration of the supernatant in this wayalso reduces the volume of liquid required to be dried to recover theprotein. The supernatant generally is concentrated to a proteinconcentration of at least about 50 g/L, preferably about 100 to about400 g/L, more preferably about 200 to about 300 g/L, prior to drying.Such concentration operation may be carried out in a batch mode or in acontinuous operation, as described above for the protein solutionconcentration step.

The concentrated supernatant then may be subjected to a diafiltrationstep using water. Such diafiltration may be effected using from about 2to about 20 volumes of diafiltration solution, preferably about 5 toabout 10 volumes of diafiltration solution. In the diafiltrationoperation, further quantities of contaminants are removed from theaqueous supernatant by passage through the membrane with the permeate.The diafiltration operation may be effected until no significant furtherquantities of phenolics and visible colour are present in the permeate.Such diafiltration may be effected using the same membrane as for theconcentration step. However, if desired, the diafiltration may beeffected using a separate membrane, such as a membrane having amolecular weight cut-off in the range of about 3,000 to about 100,000daltons, preferably about 5,000 to about 10,000 daltons, having regardto different membrane materials and configuration.

An antioxidant may be present in the diafiltration medium during atleast part of the diaflitration step. The antioxidant may be anyconvenient antioxidant, such as sodium sulfite or ascorbic acid. Thequantity of antioxidant employed in the diafiltration medium depends onthe materials employed and may vary from about 0.01 to about 1 wt %,preferably about 0.05 wt %. The antioxidant serves to inhibit oxidationof phenolics present in the concentrated canola protein isolatesolution.

In accordance with the present invention, the concentrated andoptionally diafiltered supernatant is heat treated to decrease thequantity of the 7S protein present in the solution by precipitation andremoval of the 7S protein and thereby increasing the proportion of 2Sprotein in the canola protein present in the concentrated supernatant.

Such heat treatment may be effected using a temperature and time profilesufficient to decrease the proportion of 7S present in the concentratedsupernatant, preferably to reduce the proportion of 7S protein by asignificant extent. In general, the 7S protein content of thesupernatant is reduced by at least about 50 wt %, preferably at leastabout 75 wt % by the heat treatment. In general, the heat treatment maybe effected at a temperature of about 70° to about 100° C., preferablyabout 75 to about 95° C., for about 2 to about 30 minutes, preferablyabout 5 to about 15 minutes. The precipitated 7S protein may be removedin any convenient manner, such as centrifugation or filtration.

The concentrated heat-treated supernatant, after removal of theprecipitated 7S protein, such as by centrifugation, may be dried by anyconvenient technique, such as spray drying or freeze drying, to a dryform to provide a canola protein isolate in accordance with the presentinvention. Such novel canola protein isolate has a high protein content,in excess of about 90 wt %, preferably at least about 100 wt % protein(calculated as Kjeldahl N×6.25) and is substantially undenatured (asdetermined by differential scanning calorimetry).

Such novel canola protein isolate contains a high proportion of 2Sprotein, preferably at least 90 wt % and most preferably at least about95 wt %, of the canola protein in the isolate.

In an alternative procedure to produce the novel canola protein isolate,the aqueous protein solution produced by extraction of the canola oilseed protein meal is concentrated to increase the protein concentrationthereof while maintaining the ionic strength thereof substantiallyconstant by a first ultrafiltration step using membranes, such ashollow-fibre membranes or spiral wound membranes, having a molecularweight cut-off sufficient to retain the 7S and 12S proteins in aretentate and to permit 2S protein to pass through the membrane. Asuitable molecular weight cut-off range for the membrane is from about30,000 to about 150,000 daltons, preferably about 50,000 to about100,000 daltons. For continuous operation, the membranes are dimensionedto permit the desired degree of concentration as the aqueous proteinsolution passes through the membranes.

The first ultrafiltration step may be effected to concentrate theaqueous protein solution from about 4 to about 20 fold to a proteinconcentration of at least about 50 g/L, preferably at least about 200g/L and more preferably at least about 250 g/L.

The concentrated protein solution preferably then is subjected to adiafiltration step using an aqueous salt solution of the same molarityand pH as the extraction solution. An antioxidant may be present in thediafiltration medium during at least part of the diafiltration step toinhibit oxidation of phenolics in the concentrated canola proteinisolate solution. The antioxidant may be any convenient antioxidant,such as sodium sulfite or ascorbic acid. The quantity of antioxidantemployed in the diafiltration medium depends on the material employedand may vary from about 0.01 to about 1 wt %, preferably about 0.05 wt%.

The diafiltration step may be effected by using from about 2 to about 20volumes of diafiltration solution, preferably about 5 to about 10 volumeof diafiltration solution. During the diafiltration operation, 2Sprotein phenolics and visible colour components along with other lowmolecular weight components are removed from the concentrated proteinsolution by passage through the membrane with the permeate.

The diafiltration step may be effected using the same membrane as usedfor the concentration step.

The concentration step and the diafiltration step may be effected at anyconvenient temperature, generally about 20° to about 60° C., preferablybelow about 30° C., and for a period of time to effect the desireddegree of concentration and washing. The temperatures and otherconditions used depend to some degree on the membrane equipment used toeffect the concentration and the desired protein concentration of thesolution.

The membrane used in the first ultrafiltration step permits asignificant proportion of the 2S protein to pass into the permeate,along with other low molecular weight species, including the ionicspecies of the food grade salt, carbohydrates, phenolics, pigments andanti-nutritional factors. The molecular weight cut-off is normallychosen to ensure retention of a significant proportion of the 7S and 12Sprotein in the retentate, while permitting the 2S protein andcontaminants to pass through, having regard to the different membranematerials and configurations.

The retentate from the concentration step and optional diafiltrationstep then is dried by any convenient technique, such as spray drying orfreeze drying, to a dry form. The dried protein has a high proteincontent, in excess of about 90 wt % protein, preferably at least about100 wt % protein (N×6.25), and is substantially undenatured (asdetermined by differential scanning calorimetry). The dried proteinisolate consists predominantly of the canola 7S protein, with some 12Sprotein and possibly small quantities of 2S protein. In general, thedried canola protein isolate contains:

about 60 to about 95 wt % of 7S protein

about 2 to about 15 wt % of the 12S protein

0 to about 30 wt % of the 2S protein

Preferably, the dried canola protein isolate contains:

about 70 to about 90 wt % of 7S protein

about 5 to about 10 wt % of the 12S protein

0 to about 20 wt % of the 2S protein

The permeate from the concentration step and optional diafiltration stepis concentrated in a second ultrafiltration step using membranes, suchas hollow-fibre membranes or spiral wound membranes, having a suitablemolecular weight cut-off to retain the 2S protein while permitting lowmolecular weight species, including salt, phenolics, colour componentsand anti-nutritional factors, to pass through the membrane.Ultrafiltration membranes having a molecular weight cut-off of about3,000 to about 30,000 daltons, preferably about 5,000 to about 10,000daltons, having regard to differing membrane materials andconfigurations, may be used. The permeate generally is concentrated to aprotein concentration of at least about 50 g/L, preferably about 100 toabout 400 g/L, more preferably about 200 to about 300 g/L, prior todrying. Such a concentration operation may be carried out in a batchmode or in a continuous operation, as described above for the proteinsolution concentration step.

The concentrated permeate may be subjected to a diafiltration step usingwater. An antioxidant may be present in the diafiltration medium duringat least part of the diafiltration step to inhibit oxidation ofphenolics in the concentrated permeate. The antioxidant may be anyconvenient antioxidant, such as sodium sulfite or ascorbic acid. Thequantity of antioxidant employed in the diafiltration medium depends onthe material employed and may vary from about 0.01 to about 1 wt %,preferably about 0.05 wt %.

The diafiltration step may be effected using 2 to 20 volumes ofdiafiltration solution, preferably about 5 to about 10 volumes ofdiafiltration solution. In the diafiltration operation, furtherquantities of contaminants, including phenolics and visible colourcomponents, are removed from the concentrated permeate by passagethrough the diafiltration membrane. The diafiltration operation may beeffected until no significant further quantities of phenolics andvisible colour components are removed in the permeate.

The diafiltration step may be effected using the same membrane as usedin the concentration step. Alternatively, a separate membrane may beused having a molecular weight cut-off in the range of about 3000 toabout 50,000 daltons, preferably about 5,000 to about 10,000 daltons,having regard to different membrane materials and configurations.

The concentrated and optionally diafiltered permeate is dried by anyconvenient technique, such as spray drying or freeze drying, to a dryform. The dried protein has a high protein content, in excess of about90 wt % protein, preferably at least about 100 wt % (N×6.25), and issubstantially undenatured (as determined by differential scanningcalorimetry). The dried canola protein isolate consists predominantly ofthe canola 2S protein with small quantities of 7S protein. In general,the dried canola protein isolate contains:

about 85 to about 100 wt % of 2S protein

0 to about 15 wt % of 7S protein,

preferably about 90 to about 100 wt % of 2S protein

-   -   0 to about 10 wt % of 7S protein

If desired, a portion of the concentrated canola protein isolate fromthe first ultrafiltration step may be combined with a portion of theconcentrated permeate from the second ultrafiltration step prior todrying the combined streams by any convenient technique to provide acombined canola protein isolate composition. The relative proportions ofthe proteinaceous materials mixed together may be chosen to provide aresulting canola protein isolate composition having a desired profile of2S/7S/12S proteins. Alternatively, the dried protein isolates may becombined in any desired proportion to provide any desired specific2S/7S/12S protein profile in the mixtures. The combined canola proteinisolate composition has a high protein content, in excess of about 90 wt%, preferably at least about 100 wt % (calculated as N×6.25), and issubstantially undenatured (as determined by differential scanningcalorimetry).

By operating in this manner, a number of canola protein isolates may berecovered as dry mixtures of various proportions by weight of firstultrafiltration-derived canola protein isolate and secondultrafiltration-derived canola protein isolate, generally about 5:95 toabout 95:5 by weight, which may be desirable for attaining differingfunctional and nutritional properties based on the differing proportionsof 2S/7S/12S proteins in the compositions.

DESCRIPTION OF PREFERRED EMBODIMENT

Referring to FIG. 1, there is shown therein the novel two-membraneprocess provided in accordance with one aspect of the invention incomparison to the formation of canola protein isolates (CPIs) by themicelle route.

As can be seen, retentate from a first ultrafiltration stage(Ultrafiltration #1), which may comprise ultrafiltration anddiafiltration steps, is processed in one of two ways. In thetwo-membrane process of the invention, the retentate is spray dried toprovide a canola protein isolate which consists predominantly of 7Scanola protein.

In the procedure of the aforementioned U.S. patent application Ser. No.10/137,321, the retentate is passed to a dilution step in which canolaprotein isolate is precipitated as a protein micellar mass. The proteinmicellar mass is spray dried to provide a canola protein isolateconsisting predominantly of 7S canola protein.

In the two-membrane process of the invention, the permeate from thefirst ultrafiltration step is subjected to a second ultrafiltration step(Ultrafiltration #2-A), which may include ultrafiltration anddiafiltration. The retentate from the second ultrafiltration step isspray dried to provide a canola protein isolate consisting predominantlyof 2S protein.

In the procedure of U.S. patent application Ser. No. 10/137,321, thesupernatant from the precipitation of the protein micellar mass issubjected to an ultrafiltration step Ultrafiltration #2-B), which mayinclude ultrafiltration and diafiltration. The retentate from theultrafiltration step is spray dried to provide a canola protein isolateconsisting predominantly of the 2S protein.

As described above, the retentate from the latter ultrafiltration stepmay be heat-treated to reduce the proportion of 7S protein in theretentate and to provide the novel canola protein isolate of theinvention.

EXAMPLES Example 1

This Example describes the production of a novel canola protein isolatein accordance with one embodiment of the invention.

‘a’ kg of canola meal was added to ‘b’ L of 0.1 M NaCl solution atambient temperature and agitated for 30 minutes to provide an aqueousprotein solution. The residual canola meal was removed and the resultingprotein solution was clarified by centrifugation and filtration toproduce ‘c’ L of filtered protein solution having a protein content of‘d’ % by weight.

A ‘e’ L aliquot of the protein extract solution was reduced in volume to‘f’ L by concentration on a polyvinylidene difluoride (PVDF) membranehaving a molecular weight cutoff of 5,000 daltons and then diafilteredwith ‘g’ L of 0.1 M NaCl solution on the same membrane. The diafilteredretentate was then pasteurized at 60° C. for 10 minutes. The resultingpasteurized concentrated protein solution had a protein content of ‘h’ %by weight.

The concentrated solution at ‘i’ ° C. was diluted ‘j’ into cold RO waterhaving a temperature ‘k’ ° C. A white cloud formed immediately and wasallowed to settle. The upper diluting water was removed and theprecipitated, viscous, sticky mass (PMM) was recovered from the bottomof the vessel in a yield of ‘l’ wt % of the filtered protein solution.The dried PMM derived protein was found to have a protein content of ‘m’% (N×6.25) d.b. The product was given a designation ‘n’ C300.

The parameters ‘a’ to ‘n’ for two runs are set forth in the followingTable I:

TABLE I n BW-SA034-J12-04A BW-SA035-J14-04A a 15 15 b 150 150 c 75 68 d1.93 1.95 e 75 68 f 4 4 g 20 20 h 19.08 14.20 i 33 33 j 1:10 1:10 k 3 3l 37.45 28.32 m 103.08 99.73

The removed supernatant was reduced in volume to ‘o’ L byultrafiltration using a polyethersulfone (PES) membrane having amolecular weight cut-off of 10,000 daltons and then the concentrate wasdiafiltered on the same membrane with ‘p’ L of water. The diafilteredconcentrate was then pasteurized at 60° C. for 10 minutes. Thepasteurized concentrate contained ‘q’ % protein by weight. With theadditional protein recovered from the supernatant, the overall proteinrecovery of the filtered protein solution. was ‘r’ wt %. The pasteurizedconcentrate was split into two equal portions. One portion was spraydried to form a final product given designation ‘n’ C200 and had aprotein content of ‘s’ % (N×6.25) d.b.

The parameters ‘n’ to ‘s’ for two runs are set forth in the followingTable II:

TABLE II n BW-SA034-J12-04A BW-SA035-J14-04A o 3.5 3 p 7 6 q 4.83 4.30 r49.35 38.05 s 91.73 93.69

The other portion of the pasteurized, concentrated supernatant washeated to 85° C. for 10 minutes and then centrifuged to removeprecipitated protein. The resulting concentrate was then spray dried toform a final product given designation ‘n’ C200H and had a proteincontent of ‘t’ % (N×6.25). The parameters ‘n’ and ‘t’ for two runs areset forth in the following Table III:

TABLE III n BW-SA034-J12-04A BW-SA035-J14-04A t 91.32 92.11

Example 2

This Example shows the effect of heating temperature and time on theprotein profile of canola protein isolate produced from concentratedsupernatant.

A solution of C200 canola protein isolate from batch SA035-J14-04A,prepared as described in Example 1, was prepared in reverse osmosispurified water to a protein concentration of 5 wt %. The solution wasprepared by stirring the protein and water with a magnetic stir bar forone hour at room temperature.

Samples of the protein solution (25 ml) were heated in centrifuge tubesin a temperature controlled water bath. Samples were heated for 10minutes at a temperature of 75, 80, 85, 90 or 95° C. Timing started whenthe internal temperature of the sample was within 1° C. of the desiredlevel and the samples were mixed constantly throughout the heatingprocess. After heat treatment, the samples were centrifuged at 8,000 gfor 10 minutes and the supernatants analyzed for protein profile by sizeexclusion HPLC.

TABLE IV Protein profiles of C200 solutions heated to differenttemperatures Treatment temperature %7S %2S Control (no heat) 22.6 77.475° C. 5.3 94.7 80° C. 3.6 96.4 85° C. 3.5 96.5 90° C. 1.8 98.2 95° C.1.8 98.2

As may be seen from the results set forth in Table IV, all the heattreatments applied resulted in a significant reduction in 7S proteinfrom the samples. Treatment at 90° C. resulted in the lowest level of 7Sand no additional improvement was gained by raising the temperature to95° C. It was determined by size exclusion HPLC analysis that the heattreatment had little effect on the peak area for 2S. This means that theheat treatment resulted in minimal loss of 2S protein.

A sample of protein solution (80 ml) was heated in a jacketed vesselattached to a circulating water bath set to a temperature of 90° C. andthe sample was mixed with a magnetic stir bar. Aliquots of heatedsolution were removed after 5, 10 and 15 minutes of heating time. Timingdid not start until the temperature of the sample measured 85° C. Afterheat treatment, the collected samples were centrifuged at 8,000 g for 10minutes and the supernatants submitted for analysis.

TABLE V Protein profiles of C200 solutions heated for different lengthsof time Treatment time %7S %2S Control (no heat) 22.6 77.4  5 min 1.898.2 10 min 1.9 98.1 15 min 1.9 98.1

As may be seen from the results set forth in Table V, there was nosignificant difference in the level of 7S protein in the samples for anyof the tested times.

As is apparent from the results outlined in this Example, a significantreduction in the level of 7S protein in concentrated supernatant can beobtained with a wide variety of heating conditions.

Example 3

This Example contains an evaluation of protein profiles for the canolaprotein isolates produced according to Example 1.

Size exclusion HPLC was used to evaluate the protein profile of theconcentrated supernatant and modified concentrated supernatant producedaccording to the procedures of Example 1. The spray dried products weredissolved at a 1 wt % level in 0.1 M NaCl prior to HPLC analysis.

The results obtained are set forth in the following Table VI:

TABLE VI Batch Product %12S %7S %2S SA034-J12-04A Concentrated 0.0013.13 86.67 supernatant (C200) Modified concentrated 0.00 3.55 96.45supernatant (C200H) SA035-J14-04A Concentrated supernatant 0.00 24.5275.48 (C200) Modified concentrated 000 7.55 92.45 supernatant (C200H)

As may be seen from the results set forth in Table VI, the heattreatment of the concentrated supernatant results in a significantreduction of the quantity of 7S protein in the spray dried canolaprotein isolate compared to the absence of such heat treatment.

Example 4

This Example contains an evaluation of the solubility of the canolaprotein isolates produced in Example 1.

The solubility of the spray dried concentrated supernatant (C200) andmodified concentrated supernatant (C200H) produced by the procedures ofExample 1, was determined using a modified version of the procedure ofMorr et al, J. Food Sci. 50:1715-1718.

Sufficient protein powder to supply 0.5 g of protein was weighed into abeaker and then a small amount of reverse osmosis (RO) purified waterwas added and the mixture stirred until a smooth paste formed.Additional water was then added to bring the volume to approximately 45ml. The contents of the beaker were then slowly stirred for 60 minutesusing a magnetic stirrer. The pH was determined immediately afterdispersing the protein and was adjusted to the appropriate level (4, 5,6 or 7) with NaOH or HCl. A sample was also prepared at native pH. Forthe pH adjusted samples, the pH was measured and corrected two timesduring the 60 minutes stirring. After the 60 minutes of stirring, thesample was made up to 50 ml total volume with RO water, yielding a 1%w/v protein dispersion. An aliquot of the protein dispersion wasreserved for protein content determination by Leco analysis using a LecoFA28 Nitrogen Determinator. Another portion of the sample wascentrifuged at 8000 g for 10 minutes. This sedimented any undissolvedmaterial and yielded a clear supernatant. The protein content of thesupernatant was then determined by Leco analysis.Solubility (%)=(Supernatant protein conc./Original dispersion proteinconc.)×100

The results obtained are set forth in the following Table VII:

TABLE VII Solubility (%) Batch Product pH 4 pH 5 pH 6 pH 7 Native pHSA034-J12-04A Concentrated 88.1 100 86.1 97.3 89.8 supernatant Modified100 100 100 100 100 concentrated supernatant SA035-J14-04A Concentrated87.4 95 90.6 90.6 86.7 supernatant Modified 100 100 100 100 100concentrated supernatant

As can be seen from the results of Table VII, the dried isolate frommodified concentrated supernatant (C200H) was notably more soluble inwater at various pH values than the dried isolate from concentratedsupernatant (C200).

Example 5

This Example contains an evaluation of the solubility of the canolaprotein isolates produced in Example 1 in a soft drink.

The solubility in a soft drink of the spray dried concentratedsupernatant and modified concentrated supernatant, produced by theprocedures of Example 1, were determined using a modification of theprocedure of Morr et al, J. Food Sci. 50:1715-1718.

Sufficient protein powder to supply 1.0 g of protein was weighed into abeaker and then a small amount of a colourless, transparent, commercialcarbonated soft drink was added and the mixture stirred until a smoothpaste formed. Additional soft drink was then added to bring the volumeto approximately 45 ml. The contents of the beaker were then slowlystirred for 60 minutes using a magnetic stirrer. After the 60 minutes ofstirring, the sample was made up to 50 ml total volume with soft drink,yielding a 2% w/v protein dispersion. An aliquot of the proteindispersion was reserved for protein content determination by Lecoanalysis. Another portion of the sample was centrifuged at 8,000 g for10 min. This sedimented any undissolved material and yielded a clearsupernatant. The protein content of the supernatant was then determinedby Leco analysis.Solubility (%)=(supernatant protein conc./Original dispersion proteinconc.)×100

The results obtained are set forth in the following Table VIII:

TABLE VIII Batch Product Solubility (%) SA034-J12-04A Concentratedsupernatant 92.3 Modified concentrated supernatant 94.9 SA035-J14-04AConcentrated supernatant 96.1 Modified concentrated supernatant 94.4

As may be seen from the results of Table VIII, the dried isolate frommodified concentrated supernatant (C200H) and concentrated supernatant(C200) had similar solubilities in the soft drink.

However, as may be seen from the results of Example 6 below, the clarityof the solution prepared from the dried isolate from modifiedconcentrated supernatant was far superior.

Example 6

This Example contains an evaluation of the clarity in solutions of spraydried isolates produced in Example 1 dissolved in a soft drink.

The clarity in a soft drink of solutions of spray dried isolates frommodified concentrated supernatant and concentrated supernatant, producedas described in Example 1, was determined. Clarity was assessed bymeasuring the absorbance of visible light at 600 nm by a solution of 2%w/v protein in a colourless, transparent, commercial carbonated softdrink. The lower the absorbance reading, the better light was beingtransmitted and the better the clarity of the solution.

The results obtained are set forth in the following Table IX:

TABLE IX Batch Product A600 SA034-J12-04A Concentrated supernatant 0.544Modified concentrated supernatant 0.150 SA035-J14-04A Concentratedsupernatant 1.920 Modified concentrated supernatant 0.367

As may be seen from the results of Table IX, the clarity of the solutionprepared from the spray dried isolate from modified concentratedsupernatant (C200H) was far superior to that prepared from the spraydried concentrated supernatant (C200).

Example 7

This Example illustrates an alternative process of forming the novelcanola protein isolate of the invention (FIG. 1).

15 kg of canola oil seed meal was added to 100 L (15% w/v) of 0.15 Msodium chloride solution containing 0.05 wt % ascorbic acid at ambienttemperature in a 350 L extraction tank and the mixture was agitated for30 minutes to provide a canola protein solution having a concentrationof 20 g/L. Bulk residual meal was removed by using a basket centrifugewith a 400 mesh bag and the separated bulk meal was discharged to waste.The canola protein solution was given a second pass through the basketcentrifuge using a 600 mesh bag to remove suspended fine particles. Theresulting canola protein solution was polished using a filter press with2 μm filter pads.

The clarified canola protein solution was subjected to anultrafiltration step using a spiral wound polyvinylidiene difluoride(PVDF) membrane with a molecular weight cut-off of 100,000 daltons atambient temperature to concentrate the canola protein solutioncontaining 7S and 12S proteins to a volume of 4.3 L and a proteinconcentration of 188 g/L. The permeate from the ultrafiltration stepcontained the 2S protein along with other low molecular weight species.

The concentrated canola protein solution (retentate) then was subjectedto a diafiltration step using the same membrane as for theultrafiltration using an aqueous 0.15 M sodium chloride solutioncontaining 0.05 wt % ascorbic acid. The diafiltration medium was addedto the retentate at the same flow rate as permeate was removed from themembrane. The diafiltration was carried out with 5 retentate volumes ofdiafiltration medium.

A 1.25 L aliquot of the retentate from the ultrafiltration anddiafiltration operations was spray dried to provide a canola proteinisolate consisting predominantly of 7S protein, having a protein contentof 99.1 wt % (N×6.25, percent nitrogen values were determined using aLeco FP528 Nitrogen Determinator) d.b. and containing 18.21 wt % 2Sprotein, 74.55 wt % 7S protein and 7.24 wt % 12S protein.

The permeate from the ultrafiltration and diafiltration operations wassubjected to an ultrafiltration step using a spiral woundpolyethersulfone (PES) membrane with a molecular weight cut-off of 5000daltons to permit retention of 2S protein and to permit low molecularweight contaminants to pass through the membrane to waste. Thisultrafiltration step was effected at ambient temperature to concentratethe 2S-containing permeate from the first ultrafiltration step to 3 Lhaving a protein concentration of 125 g/L.

The concentrated canola 2S protein solution (retentate) then wassubjected to a diafiltration step using the same membrane as for theultrafiltration using filtered tap water as the diafiltration medium.The water was added to the retentate at the same flow rate as permeatewas removed from the membrane. The diafiltration was carried out with 5retentate volumes of diafiltration medium.

The retentate from the diafiltration step was spray dried to provide acanola protein isolate consisting predominantly of 2S protein, having aprotein content of 105.8 wt % (N×6.25) d.b. and containing 96.7 wt % 2Sprotein, 3.3 wt % 7S protein and 0.04 wt % of 12S protein.

Example 8

This Example is a repeat of the process of Example 7, but on a largerscale.

150 kg of canola oil seed meal was added to 1000 L (15% w/v) of 0.15 Msodium chloride solution containing 0.05 wt % of ascorbic acid atambient temperature in a 10,000 L extraction tank and the mixture wasagitated for 30 minutes to provide a canola protein solution having aconcentration of 20.7 g/L. Bulk residual meal was removed by using avacuum filter belt and the separated meal was discharged to waste. Thecanola protein solution was clarified by using a disc centrifuge and thedesludged solids discharged to waste. The resulting canola proteinsolution was polished using a filter press with 2 μm filter padsfollowed by another one with 0.2 μm pads.

The clarified canola protein solution was subjected to ultrafiltrationusing two spiral wound PVDF membranes with a molecular weight cut-off of100,000 daltons to concentrate the canola protein solution containing 7Sand 12S proteins to 41.1 L having a protein concentration of 221 g/L.The permeate from the ultrafiltration step contained the 2S proteinalong with other low molecular weight species.

A 3 L aliquot of the retentate from the ultrafiltration operation wasspray dried to provide a canola protein isolate consisting predominantlyof 7S protein, having a protein content of 95.1 wt % (N×6.25) d.b. andcontaining 26.86 wt % of 2S protein, 66.22 wt % of 7S protein and 6.92wt % of 12S protein.

The permeate from the ultrafiltration operation was subjected to anultrafiltration step using two spiral wound PVDF membranes with amolecular weight cut-off of 5,000 daltons to permit retention of 2Sprotein and to permit low molecular weight contaminants to pass throughthe membrane to waste. This ultrafiltration step was effected at ambienttemperature to concentrate the 2S-containing permeate from the firstultrafiltration step to 25 L having a protein concentration of 24.2 g/L.

The retentate from the ultrafiltration step was spray dried to form anon-diafiltered canola protein product having a protein concentration of47.94 wt % (N×6.25) d.b. and containing 94.64 wt % of 2S protein, 5.36wt % of 7S protein and 0 wt % of 12S protein.

The low protein content of the latter canola protein product was due tothe absence of a diafiltration step to remove the salt and otherimpurities. Later bench diafiltration with this product given resultsthat indicated the production of a canola protein isolate afterdiafiltration.

Example 9

This Example provides a comparison of the canola protein isolateproducts prepared according to the procedure of Example 8 with canolaprotein isolate products prepared by the micelle route.

A 34 L aliquot of the retentate from the first ultrafiltration stepdescribed in Example 8 was warmed to 29.8° C. and poured into chilledwater having a temperature of 3.7° C. at a ratio of 10 volumes of waterper volume of retentate. A white cloud of protein micelles immediatelyformed. The micelles were allowed to coalesce and settle overnight. Theaccumulated protein micellar mass was separated from supernatant and wasspray dried to provide a canola protein isolate having a protein contentof 107.4 wt % (N×6.25) d.b. and containing 3.80 wt % 2S protein, 85.88wt % 7S protein and 10.32 wt % 12S protein.

The supernatant from the PMM-settling step (365 L) was subjected to anultrafiltration step using a spiral wound PVDF membrane with a molecularweight cut-off of 5000 daltons to permit retention of 2S protein and 7Sprotein and to permit low molecular weight contaminants to pass throughthe membrane to waste. This ultrafiltration step was effected at ambienttemperature to concentrate the supernatant to 22 L having a proteincontent of 89.3 g/L.

The concentrated supernatant was spray dried to provide a canola proteinisolate consisting predominantly of 2S protein, having a protein contentof 95.51 wt % (N ×6.25) d.b. and containing 84.01 wt % 2S protein, 15.51wt % 7S protein and 0.48 wt % of 12S protein.

SUMMARY OF DISCLOSURE

In summary of this disclosure, a novel canola protein isolate having anincreased content of 2S protein and a reduced quantity of 7S protein isprovided having utility in producing clear aqueous solutions,particularly in soft drinks. Modifications are possible within the scopeof the invention.

1. A process for the preparation of canola protein isolates, whichcomprises: (a) providing an aqueous canola protein solution derived fromcanola oil seed meal and containing 12S, 7S and 2S canola proteins, (b)increasing the protein concentration of the aqueous solution using aselective membrane technique which is effective to retain 7S and 12Scanola proteins in a retentate and to permit 2S protein to pass throughthe membrane as a permeate to provide a concentrated protein solution,(c) drying the retentate from step (b) to provide a canola proteinisolate consisting predominantly of 7S canola protein and having aprotein content of at least about 90 wt % (N×6.25) on a dry weight basis(d.b.), (d) increasing the concentration of the permeate from step (b)using a selective membrane technique which is effective to retain 2Scanola protein in a retentate and to permit low molecular weightcontaminants to pass through the membrane in a permeate, and (e) dryingthe retentate from step (d) to provide a canola protein isolateconsisting predominantly of 2S protein and having a protein content ofat least about 90 wt % (N×6.25) d.b.
 2. The process of claim 1 whereinsaid aqueous canola protein solution is provided by extracting canolaoil seed meal at a temperature of at least about 5° C. to causesolubilization of protein in said canola oil seed meal and to form anaqueous protein solution having a protein content of about 5 to about 40g/L and a pH of about 5 to about 6.8 and separating the aqueous proteinsolution from the residual oil seed meal.
 3. The process of claim 2wherein step (b) is effected by concentrating the aqueous solution to aprotein content of at least about 200 g/L while maintaining the ionicstrength substantially constant having an ultrafiltration membranehaving a molecular weight cut-off of about 30,000 to about 150,000daltons, preferably about 50,000 to about 100,000 daltons, to providethe concentrated protein solution.
 4. The process of claim 3 wherein theconcentrated protein solution is subjected to a diafiltration step usingabout 2 to about 20, preferably about 5 to about 10, volumes ofdiafiltration solution.
 5. The process of claim 4 wherein step (d) iseffected by increasing the concentration of the permeate to a proteinconcentration of about 100 to about 400 g/L, preferably about 200 toabout 300 g/L, using a membrane having a molecular weight cut-off ofabout 3,000 to about 30,000 daltons, preferably about 5,000 to about10,000 daltons, to provide the retentate.
 6. The process of claim 5wherein the retentate is subjected to a diafiltration step using about 2to about 20, preferably about 5 to about 10, volumes of diafiltrationsolution.
 7. The process of claim 1 wherein at least one of the canolaprotein isolates produced in steps (c) and (e) has a protein content ofat least about 100 wt %.